Construction And Fermentation Optimization Of Engineering Strain Of 3-phytosterone-9?-Hydroxylase

9?-Hydroxy-4-androstene-3,17-dione?9?-OH-AD?is one of drug intermediates for preparation of?-methasone,dexamethasone and other steroids.In industry,we often use sitosterol,stigmasterol,campesterol and etc as substrates.In general,it is obtained 9?-OH-AD by Mycobacteria and other bacteria by biotransformation.In this process,the key enzyme is 3-phytosterone-9?-hydroxylase.It is consisted of two subunits by 3-phytosterone-9?-hydroxylase?KshA?and 3-phytosterone-9?-hydroxylase reductase?KshB?.Therefore,it affects biotransformation ability of phytosterone of 3-phytosterone-9?-hydroxylase to promote the synthesis of 9?-OH-AD.This study that 3-phytosterone-9?-hydroxylase gene?KsH?is cloned and expressed in Escherichia coli and Mycobacteria,increase activity of KsH and improve the biological conversion rate of 9?-OH-AD.Firstly,primers of KshA and KshB were designed according to GenbanK gene sequence in the KsH gene,KshA and KshB gene of gene clone was obtained with Mycobacterial genomes as template by PCR.Two subunit genes was into double promoter expression vector pETDuet-1,which was transformed into E.coli BL21?DE3?,protein was expressed and verified of recombinant strains.Secondly,the vector of pNIT and KshA gene were connected,which obtained the plasmid pNIT-KshA.On the basis of pNIT-KshA,using the same method to complete the construction of pNIT-KshA-KshB,then recombinant plasmid was transformed into Mycobacterium,a positive transformants of the strain was M-ksH.The experimental results of fermentation showed that the 9?-OH-AD yield of M-ksH is higher than the original strain,which 9?-OH-AD is 0.486 g/L.In shaking flasks,fermentation media and culture conditions were also investigated through single factor experiment and orthogonal experimental design.The optimal culture media were as follows?g/L?:glycerol 20,sucrose 6,yeast powder 10,Na₂HPO₄·12H₂O 4,KH₂PO₄ 1,MgSO₄ 3,FeSO₄ 0.01,pH 8.The yield of 9?-OH-AD is 1.614 g/L,which was improved by 11.85% before optimization.